Liposome encapsulated hemoglobin

ABSTRACT

The invention relates to hemoglobin-containing liposomes in which an aqueous solution of hemoglobin is incorporated comprising liposome membrane mainly composed of hydrogenated phospholipids of hydrogenation ratio of 50% or more and an aqueous hemoglobin solution containing hemoglobin in a concentration of 30-60% (w/v). The method of preparation involves dissolving liposome membrane-forming lipids in an organic solvent, removing the solvent from said solution, adding an aqueous hemoglobin solution to the residue, subjecting the mixture to a shaking or an ultrasonication to give a uniform suspension, applying gas pressure to said suspension to allow said gas to permeate throughout said suspension and then subjecting said suspension to high pressure-delivery through fine openings. It is preferred that viscosity of the aqueous solution is 10 2  -3,000 cP (20° C.) and the inert gas pressure is 80-130 kg/cm 2 .

TECHNICAL FIELD

The present invention relates to a novel method for preparing liposomes.More particularly, the invention is concerned with a method forpreparing liposomes with an aqueous solution innerly incorporated.

Furthermore, the invention relates to novel hemoglobin-containingliposomes (called hemosomes herein below) with a highly concentratedaqueous solution of hemoglobin innerly contained.

Liposomes are closed vesicles consisting of lipid bilayers with aqueousspaces innerly contained. Biological membranes are believed to be oflipid bimolecular structure. In this respect, liposomes are widely usedin the study on physicochemical properties of the biomembrane as a modelmembrane. Various substances can be incorporated in aqueous spaces or inphospholipid bilayers of liposomes. The liposomes are fused with orincorporated in the cells so that they are also used as a carrier fordelivering a substance into the living body. Studies using liposomescover a wide variety of fields including biology, medical science andpharmaceutical science. Studies have been made on its application as acarrier for delivering oxygen or an anticancer drug, immunologicalapplication, cell interaction or application as a drug delivery system,etc.

Moreover, hemosomes are expected to be useful as artificial erythrocyteshaving high oxygen carrying capacity and safety and being stable tooxidation.

BACKGROUND TECHNOLOGY

As described above, liposomes are utilized in a wide variety of fields.However, it was impossible by the prior-art methods for preparingliposomes to produce liposomes with a high-viscosity aqueous solutioninnerly incorporated. Prior methods for preparing liposomes includeso-called film methods, detergent-removal methods and reverse-phaseevaporation methods. The film methods comprises forming a dried thinfilm of liposome-forming lipids on the inner surface of a vessel, towhich an aqueous solution of a substance to be incorporated is added andsubjecting the resulting mass to shaking or ultrasonication. Thedetergent-removal method depends upon removing detergents by dialysisfrom an aqueous solution which contains detergents and phospholipids toform mixed micelles, which results in the formation of liposomes. Thereverse-phase evaporation method is a method in which liposomes areprepared by adding to an organic solvent solution of liposome-forminglipids an aqueous solution of a substance to be incorporated to form awater-in-oil emulsion and then removing the organic solvent byevaporation. According to these prior art methods, whereas liposomes areformed in cases where the aqueous solution to be innerly incorporated isof a low viscosity, the yield will be extremely low if it is of a highviscosity over 10 cP (20° C.), and desirable liposomes will not beproduced in some cases. This has restricted uses of liposomes. Forexample, liposomes containing an aqueous hemoglobin solution are knownas artificial erythrocytes, but, because of the viscosity restriction,the hemoglobin concentration cannot be so high as that of naturalhemoglobin (which is 35% (w/v)), being as low as approximately 15%, sothat the oxygen-carrying capacity is low.

On the other hand, Miller et al. reported a hemoglobin-containingliposome prepared by the so-called film method (U.S. Pat. No.4,133,874). According to the method, the liposome is produced bydissolving liposome-forming lipids in an appropriate solvent such aschloroform, distilling off the solvent from the resulting solution toform a film of the lipids, to which an aqueous solution of hemoglobin isadded and then subjecting the liposome to ultrasonication. The method isadvantageous in that the hemoglobin can be kept with relatively littledegradation due to contact with oxygen only for a short period. However,the oxygen-carrying function is likely to be lost due to slow oxidationof the heme iron of hemoglobin in the liposome during storage. Althoughhemoglobin in the blood cell is provided with a mechanism wherein thehemoglobin oxidized to the methemoglobin is reduced to the original formby the action of enzymes, such mechanism is not workable if removedoutside the blood cell by hemolysis so that the oxygen-carrying capacityis lost once denatured to the methemoglobin.

Hemoglobin is a macromolecular substance having a molecular weight ofabout 65,000 the aqueous solution of which has a high viscosity.According to the prior methods liposomes containing an aqueous solutionwith such a high molecular weight and viscosity cannot be prepared, andthere are obtained liposomes containing hemoglobin in concentration aslow as about 15%. As compared with the hemoglobin concentration of about35% in natural erythrocytes, the above-cited concentration is not highenough for carrying oxygen. In order to provide a oxygen-carryingcapacity equal to that of blood, the concentration of the hemosomeitself cannot be increased, which will increase the concentration of themembrane-forming lipid material and induce a safety problem.Furthermore, viscosity of the lipid suspension will become so high thatthe dynamics blood flow will unfavorably be affected. Under suchcircumstances, liposomes innerly containing hemoglobin in a higherconcentration are desirable.

The above-mentioned object is achieved by the present invention adescribed below.

SUMMARY OF THE INVENTION

(1) A method for preparing liposomes which comprises dissolving liposomemembrane-forming lipids in an organic solvent, removing the solvent fromsaid solution, adding an aqueous solution to the residue, subjecting themixture to shaking or ultrasonication to give an uniform suspension,applying gas pressure to said suspension to allow said gas to permeatethroughout said suspension and then subjecting said suspension to highpressure-delivery through fine openings.

(2) A method according to item 1 wherein viscosity of the aqueoussolution is 10-3,000 cP (20° C.).

(3) A method according to item 1 wherein the gas is an inert gas.

(4) A method according to item 1 wherein the inert gas is applied at apressure of 80-130 kg/cm².

(5) A method according to item 3 wherein the suspension is subjected toa high pressure-delivery treatment using a gas-pressurizing highpressure-delivery emulsifier.

(6) A hemoglobin-containing liposome in which an aqueous solution ofhemoglobin is incorporated comprising a liposome membrane mainlycomposed of hydrogenated phospholipids having a hydrogenation percentageof 50% or more and an aqueous hemoglobin solution containing hemoglobinin a concentration of 30-60% (w/v).

(7) A hemoglobin-containing liposome according to item 6 wherein thehydrogenated phospholipids are hydrogenated natural phospholipids.

(8) A hemoglobin-containing liposome according to item 7 wherein thehydrogenated natural phospholipids are hydrogenated lecithins.

(9) A hemoglobin-containing liposome according to item, 8 comprisinghydrogenated lecithin having a hydrogenation percentage of 80% or more.

(10) A hemoglobin-containing liposome according to item 9 comprisinghydrogenated phosphatidylcholine having a percentage of hydrogenation of80% or more.

(11) A hemoglobin-containing liposome according to item 6 wherein thehemoglobin concentration is 50% (w/v).

(12) A hemoglobin-containing liposome according to any of the items 6-10wherein the liposome membrane contains a negative charge-providingsubstance.

(13) A hemoglobin-containing liposome according to item 12 wherein thenegative charge-providing substance is phosphatidic acid, dicetylphosphate or a higher fatty acid.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graph indicating change with time of methemoglobin ratio (%)in the hemosomes.

FIG. 2 is a graph indicating viscosity of the hemosome suspension.

DETAILED DESCRIPTION OF THE INVENTION

There is no particular limitation on the liposome membrane-forminglipids, and any natural or synthetic lipids that form liposomes may beused. Especially preferred are phospholipids. As examples are mentionedlecithins, phosphatidylethanolamine, phosphatidic acid,phosphatidylserine, phosphatidylinositol, phosphatidylglycerol,sphingomyelin, cardiolipin and their hydrogenation products prepared bya conventional method. Their combination may also be employed. To theliposome membrane constituent may be added a membranestructure-reinforcing agent such as sterols and a disintegrationtime-controlling agent of an electric charge-providing substance (forexample, stearic acid, oleic acid, linolic acid, linoleic acid,phosphatidic acid and phosphatidylglycerol). There is no particularlimitation on the high viscosity-aqueous solution to be innerlyincorporated in liposomes, and an aqueous solution of any chemicalsubstance may be used. As examples of the chemical substance arementioned, in addition to hemoglobin as described above, macromolecularcompounds such as 8-glucuronidase, heparinase and β-glucosidase.

The viscosity of the aqueous solution is determined in accordance withthe use of liposomes and depending upon nature of the solute. An aqueoussolution having a viscosity in the range of 10 cP-3,000 cP (20° C.) isusually employed in the invention. Although an aqueous solution having aviscosity of 10 cP or below (20° C.) may be used in the invention, itcan also be used in the prior-art preparative methods. For preparationof artificial erythrocytes, an aqueous solution of hemoglobin in aconcentration of 30-60% (w/v) is preferably employed with the viscosityof 10-3,000 cP (20° C.).

The liposomes of the invention are prepared by dissolving liposomemembrane-forming lipids in an organic solvent, removing the solvent fromsaid solution, adding an aqueous solution to the residue, subjecting themixture to shaking or ultrasonication to give a uniform suspension,applying gas pressure to said suspension to allow said gas to permeatethroughout said suspension and then subjecting said suspension to highpressure-delivery through fine openings.

The steps up to preparation of an uniform suspension in theabove-described method is the same as in the previously-mentioned methodfor preparing liposomes known as the film method, and are carried out byprocedures known per se. The organic solvent is selected for specificapplications of liposomes, and is usually chloroform, ethanol or thelike.

It is preferable to use an inert gas such as nitrogen or argon as thegas used for pressure application when the aqueous solution contains asubstance likely to degrade or when hydrogenation of the phospholipidsis low.

Pressure is applied to the suspension thus obtained in a pressure vesselequipped with a nozzle of fine openings by an inert gas (for example,nitrogen or argon). Adequate pressure is 80-130 kg/cm² After the inertgas has been thoroughly permeated through the suspension, the suspensionis subjected to a delivery treatment from the nozzles.

The step for high pressure-delivery treatment of the suspension iscarried out by delivering the suspension once or several times throughthe openings by means of a high pressure delivery emulsifier, preferablya gas-pressurizing high pressure-delivery emulsifier while maintainingthe above-mentioned pressure. In this step, the suspension through whichthe inert gas has been permeated is vigorously emulsified by rapidexpansion of the gas under pressure to form liposomes. Higher pressuredelivery produces liposomes of smaller particle sizes. Unheld materialsare washed out of the liposomes thus obtained by a conventional method,and the liposomes are isolated by ultracentrifugal treatment or thelike.

It is noted that the gas-pressurizing high pressure-delivery emulsifierused in the invention comprises an usual gas bomb or a high pressure gassupplier and a pressure vessel equipped with a fine-opening nozzle, thepressure vessel being provided in communication with said gas bomb orhigh pressure gas supplier. Since the principle is simple, the methodcan be widely applied to various scales from a small production toindustrial one by using a pressure vessel corresponding to the amount tobe treated. Also, the use of few mechanical parts are advantageouslyassociated with little troubles.

The membrane material of the hemosomes according to the invention ismainly composed of hydrogenated phospholipids having a percentage ofhydrogenation of 50% or more.

The fatty acid composition of natural phospholipids, obtained fromsoybean, egg yolk and others contain a lot of unsaturated fatty acidchains. In the present invention, hydrogenated phospholipids in which50% or more of unsaturated fatty acid chains of the naturalphospholipid, as mentioned above, being saturated with hydrogen arepreferably used.

Synthetic distearoyllecithin is stable to oxidation due to the absenceof unsaturated fatty acids in the molecule but is inferior in thetrapping of hemoglobin compared to hydrogenated natural lecithins. It isbelieved that while hydrogenated phospholipids are a mixture ofphospholipids containing in the molecule various fatty acids, syntheticdistearoyllecithin is a single compound containing as the fatty acidstearic acid alone.

The percentage of hydrogenation in the hydrogenated phospholipids usedin the invention is 50% or more, preferably 80% or more and is, in termsof the iodine value, not more than 30, preferably not more than 10. Apercentage of hydrogenation less than 50% will be insufficient for theprevention of hemoglobin oxidation. As examples of the hydrogenatedphospholipid are mentioned hydrogenation products of lecithins,phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine,phosphatidylglycerol, sphingomyelin, cardiolipin and the like producedby a conventional method. Particularly preferred are hydrogenatednatural lecithins produced by hydrogenation of soybean lecithin, eggyolk lecithin, corn lecithin, cotton seed oil lecithin, rape oillecithin, etc.

The percentage of hydrogenation is determined as follows: The fatty acidchains of hydrogenated phospholipids are converted to their methylesters by the method of Jhum et al. (J. Am. Oil Chem. Soc. 53, 132(1982)]and then analyzed by GLC for calculation of the iodine value fromthe composition of fatty acids. The iodine value of the unhydrogenatedphospholipid is determined in the same way as above, and the percentageof hydrogenation is calculated from the percent of the latter to theformer iodine value of hydrogenated phospholipids.

To the membrane material of the hemosomes according to the invention maybe added a sterol such as cholesterol or cholestane in order toreinforce the membrane. Also in order to prevent aggregation of theliposome a negative charge-providing substance, for example,phosphatidic acid, a higher fatty acid or dicetyl phosphate may beadded. In addition, when an antioxidant such as tocopherol (vitamin E)is added to the membrane material, oxidation of the liposome membrane isinhibited.

For example, tocopherol may be added in a proportion of 0.01-0.1 mol,preferably 0.04 mol per mol of phospholipids.

As the hemoglobin to be innerly incorporated in the liposomes is used aproduct obtained by hemolyzing erythrocytes according to a conventionalmethod followed by ultrafiltration using a membrane for fractionation ofmolecular weights of 50,000 and below to a concentration of 30% (w/v) orhigher. Hemoglobin is incorporated into liposomes in an aqueous solutionin a concentration of 30-60% (w/v), the viscosity being approximately10-3,000 cP (20° C.).

The hemosomes of the invention are prepared by dissolving a liposomemembrane material mainly composed of the above-described hydrogenatednatural phospholipid in an organic solvent, removing the solvent fromsaid solution, adding an aqueous solution of hemoglobin in aconcentration of 30-60% (w/v) to the residue, subjecting the mixture toshaking or ultrasonication to give on uniform suspension, applyingpressure by inert gas to said suspension to allow the inert gas topermeate throughout said suspension and then subjecting said suspensionto high pressure-delivery through fine openings.

Steps up to the preparation of the homogeneous suspension in theabove-described preparative method is the same as in theliposome-preparative method known as the film method as mentioned aboveexcept for the use of a membrane material mainly composed ofhydrogenated natural phospholipids. These steps are carried out byprocedures known per se. As the organic solvent are usually employedchloroform, ethanol and the like.

The suspension thus obtained is treated by the above-described highpressure-delivery method, which is washed by a conventional method andsubjected to an ultracentrifugal treatment to afford the desirablehemosomes.

The invention will be described in more detail with reference toexamples and test examples.

EXAMPLE

(1) Preparation of stroma-free hemoglobin (SFH) solution

Using a blood-collecting pack containing an anticoagulant, 15 lit. ofwhole bovine blood is collected from the vein. The collected whole bloodis aseptically transported and stored in a closed vessel at 4° C. All ofthe subsequent steps are carried out aseptically at a low temperature.

Centrifugal washing is effected with a physiological salt solution bymeans of a continuous centrifuge thereby producing 5 lit. of crudewashed erythrocytes in which platelets, leucocytes and plasma have beenremoved.

Further washing is carried out with physiological salt solution by meansof a plasma separator 0.45 μ in pore diameter. Hemolysis is attained byusing 10 lit. of pyrogen-free distilled water per 5 lit. of the washederythrocytes. Removal of the erythrocyte membrane and filtrationsterilization are conducted by means of a plasma separator 0.45 μm indiameter and a plasma component separator 0.1 μm in diameter,respectively. There is obtained approximately 12 lit. of stroma-freehemoglobin in a hemoglobin concentration of 8% (w/v).

Ultrafiltration using a dialyzer for dialysis TAF10W (a cellulose hollowdialyzer manufactured by Terumo Corporation) gives approximately 1.8lit. of stroma-free hemoglobin (SFH) solution in a hemoglobinconcentration of 50% (w/v).

(2) Preparation of liposome-forming lipids

In chloroform are dissolved 27.76 g of purified egg yolkphosphatidylcholine having a percentage of hydrogenation of 80%, 6.96 gcholesterol and 3.75 g of purified phosphatidic acid having a percentageof hydrogenation of 80%. Said lipid solution is placed in a round-bottomflask and subjected to evaporation to remove the chloroform, therebyforming the lipid membrane at the bottom of the flask. Further, vacuumdrying is conducted for 16 hours to completely remove the chloroform.

(3) Preparation of SFH-lipid mixture

To said lipid membrane is added 300 ml of the 50% SFH obtained in theabove SFH preparative step. The mixture is subjected to shaking orultrasonication to give a uniform suspension, which is used as thestarting material.

(4) Preparation of hemoglobin-containing liposomes by pressure deliverywith inert gas

Said starting material is placed in a Parr Cell Disruption Bomb(manufactured by Parr Instrument Company, USA) which is a pressurevessel with a fine-opening nozzle, and pressurized at 130 kg/cm² byintroducing nitrogen gas. The nitrogen gas is thoroughly permeatedthrough the starting material by standing for 30 min. Then the valve ofthe nozzle is slowly opened to deliver the starting material whilemaintaining the pressure at 130 kg/cm².

(5) Purification of hemoglobin-containing liposomes

The material after the delivery is ten times diluted with physiologicalsalt solution and centrifuged (17,000 rpm, 30 min.) to separatehemoglobin-containing liposome precipitates. The hemoglobin in thesupernatant that has not participated in the capsulation is removed bydecantation. Then, the hemoglobin-containing liposome precipitates aresuspended in a physiological salt solution, and the suspension is againcentrifuged. The same procedures are repeated until no hemoglobin isdetected in the supernatant.

(6) Removal of coarse particles

The suspension of the hemoglobin-containing liposomes after thepurification is filtered through a porous polycarbonate filter 0.4 μ inpore diameter (manufactured by Nuclepore Corporation, USA) to removecoarse particles.

(7) Adjustment of hemoglobin concentration

There was obtained 180 ml of a suspension in a physiological saltsolution adjusted to a hemoglobin concentration of 10%.

Yield of the hemoglobin at this stage was ##EQU1##

TEST EXAMPLE 1 Oxidation of hemoglobin

The hydrogenated phospholipid hemosomes prepared in Example (No. 1),non-hydrogenated phospholipid hemosomes (containing vitamin E) (No. 2)and non-hydrogenated phospholipid hemosomes (containing no vitamin E)(No. 3) were respectively suspended in a physiological salt solution andallowed to stand at 37° C. Ratio of methemoglobin (%) was then measuredwith the passage of time.

Results are shown in FIG. 1. As clearly seen in FIG. 1, the oxidation(metho conversion) preventive effect is observed with the hydrogenatedphospholipid hemosomes of the invention (No. 1).

Test Example 2

The lipid concentration-decreasing effect of the conversion of aconcentrated aqueous solution of hemoglobin to liposomes on hemoglobinconcentration in the hemosome suspension was examined.

Ratio of lipid concentration in the hemosome suspension: L (mg/ml) tohemoglobin concentration: H (mg/ml) in the 50% SFH hemosome prepared inExample (No. 1), a hemosome prepared from 30% (w/v) SFH (No. 4) and ahemosome prepared from 15% (w/v) SFH (No. 5) respectively as control,namely, L/H is shown in Table 1.

                  TABLE 1                                                         ______________________________________                                                  Sample       L/H                                                    ______________________________________                                        No. 1       50% SFH hemosomes                                                                            1.29                                               No. 4       30% SFH hemosomes                                                                            3.48                                               No. 5       15% SFH hemosomes                                                                            6.76                                               ______________________________________                                    

As shown in Table 1, the effect of decrease in lipid concentration on Hbconcentration is clearer as SFH concentration increases.

TEST EXAMPLE 3

Effect of the conversion of a concentrated aqueous hemoglobin solutionto liposomes on decrease in the viscosity of a hemosome suspension wasexamined.

The viscosity of 50% SFH hemosome suspension (No. 1), SFH hemosomesuspension (No. 4) and 15% SFH hemosome suspension (No. 5),respectively, were examined when hemoglobin concentration in thehemosome suspension (physiological salt solution) was kept constant(7%). Results are shown in FIG. 2.

It is clear from FIG. 2 that when the viscosity is compared at aconstant hemoglobin concentration in the hemosome suspension, the higherthe content of SFH the greater decrease in the viscosity of the hemosomesuspension.

COMPARATIVE EXAMPLE

The SFH-lipid suspension prepared in Example (Stage 3 in the example)was treated with a French press (Ohtake Seisakusho) The same effect asin the Example was searched with reference to the particle size. Whereasone treatment under a pressure of 200 kg/cm² formed liposomes, theparticle size was irregular. Five treatments were needed in order toproduce the same particle size as in the Example. The same procedures asin and after Stage 5 in the Example also afforded 154.5 ml of artificialerythrocytes. The yield was

    [10×154.5 (ml) / 50×300 (ml)]×100 (%)=10.3%,

which yield was also inferior as compared with the Example. Operationtime was also found shorter in the Example.

What is claimed is:
 1. A hemoglobin-containing liposome in which anaqueous solution of hemoglobin is incorporated comprising a liposomemembrane composed of a hydrogenated phopholipid having a percentage ofhydrogenation of 50% or more and an aqueous hemoglobin solutioncontaining hemoglobin in a concentration of 30-60% (w/v).
 2. Ahemoglobin-containing liposome according to claim 1 wherein thehydrogenated natural phospholipids are hydrogenated lecithin.
 3. Ahemoglobin containing liposome according to claim 2 comprisinghydrogenated lecithin having a percentage of hydrogenation of 80% ormore.
 4. A hemoglobin-containing liposome according to claim 3comprising hydrogenated phosphatidylcholine having a percentage ofhydrogenation of 80% or more.
 5. A hemoglobin-containing liposomeaccording to claim 1, wherein the hemoglobin concentration is 50% (w/v).6. A hemoglobin-containing liposome according to claim 5 wherein thenegative charge-providing substance is phosphatidic acid or dicetylphosphate.
 7. A hemoglobin-containing liposome in which an aqueoussolution of hemoglobin is incorporated comprising a liposome membranecomposed of a hydrogenated phospholipid having 50% or more hydrogenationand an aqueous hemoglobin solution containing hemoglobin in aconcentration of 30-60% (w/v) prepared by dissolving liposomemembrane-forming lipids in an organic solvent, removing the solvent formsaid solution thereby leaving a residue, adding an aqueous solution tothe residue, subjecting the mixture to shaking or ultrasonication togive an uniform suspension, applying gas pressure to said suspension toallow said gas to permeate throughout said suspension and thensubjecting said suspension to high pressure-delivery through fineopenings.
 8. A hemoglobin-containing liposome according to claim 7wherein viscosity of the aqueous solution is 10-3,000 cP (20° C.).
 9. Ahemoglobin-containing liposome according to claim 7 wherein the gas isan inert gas.
 10. A hemoglobin-containing liposome according to claim 7wherein the inert gas is applied at a pressure of 80-130 kg/cm².
 11. Ahemoglobin-containing liposome according to claim 7 wherein thesuspension is subjected to a high pressure-delivery treatment using agas-pressurizing high pressure-delivery emulsifier.